Ethylene is one of five well-established plant hormones. It plays an important role in virtually every phase of plant development including seed germination, fruit ripening, leaf and flower senescence, and abscission. The production of ethylene may also be induced by external factors such as mechanical wounding, anaerobiosis, auxin treatment, ultraviolet light, temperature extremes, water stress, and ions such as cadmium, and lithium (Abeles, F. B., 1973, Ethylene in Plant Biology, 197-219, Academic Press, London; Yang & Hoffman, 1984, Annu. Rev. Plant Physiol., 35, 155-189).
The pathway for ethylene biosynthesis has been established, the first step of which involves the formation of S-adenosyl-L-methionine (AdoMet) by S-adenosyl-L-methionine synthetase. AdoMet is subsequently converted by S-adenosyl-L-methionine methylthio-adenosine-lyase (ACC synthase; EC 4.4.1.14) to the nonprotein amino acid 1-aminocyclopropane-1 carboxylic acid (ACC), the immediate precursor of ethylene in higher plants (Adams & Yang, 1979, Proc. Natl. Acad. Sci. USA, 76, 170-174). Physiological analysis has suggested that this is the key regulatory step in the pathway, (Kende, 1989, Plant Physiol., 91, 1-4). Thus, the rate of endogenous expression of ACC synthase is considered to limit substantially the rate of ethylene production.
It is well known that endogenous ethylene is often deleterious to crops. In particular, increased ethylene production due to trauma caused by mechanical wounding of fruits and vegetables, and the cutting of flowers greatly diminishes their post harvest quality and storage life. However, the role of ethylene in regard to initiation of flowering in plants is not so clear. In this respect, ethylene is known to inhibit flowering in most plant species but in regard to mango and species of Bromeliad including pineapple, ethylene has been shown to promote initiation of flowering (Salisbury and Ross, 1992, Plant Physiology, Wadsworth Inc., California, p. 682). Accordingly, ethylene, ethylene producing compounds and auxins have been used almost universally to artificially induce flowering in commercial pineapple production (Turnbull et al, 1993, Acta Horticulturae, 334, 83-92).
However, flower initiation in pineapple can occur naturally once a minimum plant size and age is attained (Py et al, 1987, "The Cultivation of Pineapple", G. P. Maisonneuve Et Larose, Paris, p. 567; Bartholomew and Malezieux, 1994 In Schaffer and Anderson, 1994, "Handbook of Environmental Physiology of Fruit Crops, CRC Press, Florida, p. 310). This generally causes problems because if natural flower initiation occurs in a portion of the crop before the application of ethylene producing compounds thereto, crop control will only be partial.
In many countries, natural flowering of pineapple plants is a major industry problem because a substantial portion of a particular crop flowers naturally at different times in comparison to the remainder of the crop which has been induced artificially. For example, in Australia, natural initiation of flowering usually causes 5 to 40% of a summer crop to produce fruit which matures 4 to 6 weeks ahead of the normal summer harvest. This increases the number of passes required to pick that crop and generally results in some fruit being over-ripe. These factors decrease substantially the profits associated with growing a pineapple crop. Furthermore, cost benefits that would be possible if crop harvests could be planned completely cannot be achieved because a grower must artificially induce a crop at times designed to circumvent natural initiations.
Accordingly, the control of flowering to thereby control the timing and spread of fruit ripening within a crop is an important industry objective. If flowering and hence the uniformity of fruit maturity within the crop can be better controlled, fewer passes will be necessary to pick the fruit, and less fruit will be lost as a consequence of over-ripeness or immaturity. Furthermore, the control of flowering and hence fruit maturity will assist substantially in the organisation of labour associated with harvesting and processing activities and allow more flexibility in planning crop schedules (Py et al, 1987, supra).
At the present time, there are no satisfactory means for the prevention of natural flowering in pineapple plants. However, a strategy has been developed recently that takes advantage of the modulation properties of ACC synthase in the control of ethylene biosynthesis. In this regard, reference may be made to International Application No. PCT/US91/06453 which is directed to inhibition of expression of endogenous ACC synthase using an antisense expression system. This system comprises a DNA molecule capable of generating, when contained in a plant host cell, a complementary RNA that is sufficiently complementary to an RNA transcribed from an endogenous ACC synthase gene to prevent the synthesis of endogenous ACC synthase. Ethylene production in fruits of transgenic tomato plants engineered using this system was inhibited by 99.5% and, as a consequence, fruit ripening was suppressed. In addition, the application of ethylene or propylene to the fruits of these plants restored normal ripening.
Thus, ACC synthase genes may be used as targets for the generation of transgenic plants in which endogenous expression of ACC synthase is inhibited to effect suppression of ethylene production. The efficacy of this system, however, is predicated on the condition that the antisense RNA is sufficiently complementary to the transcript expressed from the target gene. In this regard, many studies have shown that ACC synthase is encoded by a highly divergent multigene family (for a review, see Theologis, A. 1992, Cell, 70, 181-184; Kende, H., 1993, Annu. Rev. Plant Physiol. Plant Mol. Biol., 44, 283-307). Accordingly, if there is diversity between different ACC synthase genes, the use in this system for example, of a particular ACC synthase gene from one plant species would not be expected to inhibit the expression of an ACC synthase gene from another plant species. This is supported on page 5 of PCT/US91/06453 which states that "[w]hile the various ACC synthases are generally active in a variety of plant tissues, the DNAs are not completely homologous, and therefore the use of the genetic materials for control of synthesis, for example, using an antisense strategy, does not translate cross species."
In addition to sequence diversity of ACC synthases across species, it is well known that there is substantial sequence diversity of ACC synthases encoded within species. For example, in tomato, ACC synthase is encoded by six genes, two of which are expressed in fruit ripening (Van der Straeten et al., 1990, Proc. Natl. Acad. Sci. USA, 87, 4859-4863; Olson et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 5340-5344; Rottmann et al., 1991, J. Mol. Biol., 222, 937-961; Yipp et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 2475-2479). The other four ACC synthases have been purported to be involved in other stages of plant development.
Reference also may be made to an article by Sato and Theologis (1989, Proc. Natl. Acad. Sci. USA, 86, 6621-6625) which describes the presence of a number of homologous but different ACC synthases in zucchini. In this regard, the various ACC synthases were shown to control ethylene production during different developmental processes, thus permitting separate control of, for example, fruit ripening and seed germination.
Accordingly, if there is diversity between different ACC synthase genes within plant species, the use, for example, of a particular ACC synthase gene involved in one stage of plant development in the antisense system of PCT/US91/06453 would not be expected necessarily to inhibit the expression of an ACC synthase gene involved in another stage of plant development.